Genetic modification of hematopoietic stem cells to provide chemoprotection and allow for in vivo selection has tremendous potential for the treatment of malignant and nonmalignant diseases. Unfortunately, stem cell gene therapy and in vivo selection strategies developed in the mouse have generally not been predictive for outcomes in large animal or human studies. Thus, we propose to study in vivo selection strategies in the dog, a well-established clinically relevant large animal model for stem cell gene therapy and transplantation. We propose to use O6-benzylguanine (BG) resistant mutants of O6-methylguanine-DNA methyltransferase (MGMT) as selectable drug-resistance genes in combination with BCNU and temozolomide. Our first objective is to determine the optimal vector and MGMT mutant for efficient chemoprotection and selection of MGMT-transduced cells in the autologous transplant setting. Results from these studies will be directly relevant for ongoing stem cell gene therapy protocols in patients with genetic diseases and for patients with malignant diseases receiving chemotherapy with BCNU or temozolomide. The second object of this application is to extend in vivo selection strategies to the nonmyeloablative allogeneic transplantation setting. Clinical studies have shown that high-dose chemotherapy is not required for engraftment of allogeneic stem cells, and nonmyeloablative transplantations are now being performed for many malignant and nonmalignant diseases. However, graft rejection, relapse, and GVHD have remained significant problems after nonmyeloablative transplantation. Here we hypothesize that gene-modified, chemoprotected allogeneic stem cells can be used to a) permit safer and less toxic administration of post-transplant chemotherapy in the setting of persistent or recurrent disease and b) facilitate engraftment of allogeneic stem cells after nonmyeloablative transplantation. Our preliminary data indicate that the dog is an excellent preclinical model to study the feasibility of combining stem cell gene therapy with nonmyeloablative transplantation, two highly powerful treatment strategies. These experiments will provide a platform for studies using gene-modified allogeneic stem cells to improve nonmyeloablative transplant strategies.